Mitochondrially Targeted PARP Inhibitor, and Uses Thereof

ABSTRACT

A mitochondrial-targeted PARP inhibitor is provided herein, as well as methods of making and using the mitochondrial-targeted PARP inhibitor.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/340,237 filed Apr. 8, 2019, which is a United States National Phaseof International Application No. PCT/US2017/056503 filed Oct. 13, 2017,which claims the benefit of U.S. Patent Application No. 62/407,639 filedOct. 13, 2016, each of which is incorporated herein by reference in itsentirety.

Poly(ADP-Ribose) Polymerase (PARP) is a family of enzymes involved inDNA repair, genome stability, cellular energy metabolism and celldivision. Poly(ADP-ribose) polymerases (PARP) are abundant cellularenzymes, with PARP-1 being the most well characterized PARP familymember. PARP enzymes build poly(ADP-ribose) polymers (PADPRp) ontotarget proteins including histones and PARP-1 itself, converting NAD₊ toADP-ribose and nicotinamide while consuming cellular NAD₊ stores in theprocess. PARP-1 is activated by DNA damage and plays a key role ingenomic DNA repair, reflected by the fact that it is the second mostabundant protein in the cell's nucleus. However, over-activation ofPARP-1 can result in critical depletion of cellular NAD₊ and initiationof mitochondrial-triggered cell death cascades, leading the scientificfield to coin the phrase “suicide theory of PARP activation” (Szabo etal. Trends Pharmacol Sci 1998; 19:287-98) and the term “PARthanatosis”(Andrabi et al. Ann N Y Acad Sci 2008; 1147:233-41), respectively, tocharacterize PARP-mediated mechanisms of cell death.

Poly(ADP-ribosyl)ation (PARylation) plays a central role in cellular andmolecular processes including DNA damage detection and repair,transcription, and the maintenance of genomic integrity. Thecurrently-identified 17 members of the poly(ADP-ribose) polymerase(PARP) family induce the cleavage of NAD⁺ into nicotinamide andADP-ribose moieties and mediate their polymerization on target proteins,with links to cellular redox homeostasis, inflammatory, and metabolicnetworks. While there are broader therapeutic implications for syntheticPARP modulators, poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors havebecome a hot topic in cancer research since the regulatory approval ofolaparib for patients with BRCA1/2 mutant ovarian cancer. PARP-1 bindsto nuclear DNA single-strand break (SSBs) sites and recruits repairproteins to the DNA, subsequently dissociating itself from the SSB. Themost potent small-molecule inhibitors, rather than just off-settingPARP-1's enzymatic activity, trap it at the SSB site and stabilizePARP-DNA complexes, ultimately causing DNA double-strand breaks thatrequire more complex repair mechanisms.

DNA damage induced by irradiation or oxidative stress leads toover-activation of PARP-1 and induces depletion of cellular NAD⁺ and ATPlevels, leading to cell dysfunction and necrotic cell death. A primarylocation of NAD⁺ is in mitochondria, where it is utilized for oxidativephosphorylation. Furthermore, mitochondrial DNA (mtDNA) is constantlybeing exposed to damaging species such as reactive oxygen and nitrogenspecies and is efficiently repaired through at least a subset of thespecies involved in nuclear DNA repair, including PARP-1. In addition tosome cancers being exquisitely susceptible to NAD⁺ depletionover-activation of PARP-1 and NAD⁺ depletion has been linked to thepathogenesis of central nervous system (CNS) disorders, includingischemia, traumatic brain injury (TBI), neuroinflammation, andneurodegenerative diseases such as Alzheimer's and Parkinson's diseasesand chronic traumatic encephalopathy, which have a pronouncedmitochondrial component.

It is a widely held belief that PARP activation within cell nucleiaccounts for the entirety of PARP-mediated cell death. As such, all PARPinhibitors developed to date target nuclear PARP activation.Disappointingly, despite numerous promising pre-clinical studies datingback to the 1990's targeting nuclear PARP overactivation to prevent celldeath, there are no successful clinical applications using PARPinhibitors as mitigators of cell death. Not only that, but PARPinhibitors are being evaluated in cancer trials to enhance tumoricidalactivity of DNA damaging chemotherapeutic agents by preventing DNArepair in cancer cells.

Inhibition of PARP-1, a well-characterized member of this family, hasbeen explored as a strategy for enhancing anti-cancer activity ofexisting drugs and for developing new drugs. Recently unique enzymaticproperties and biological functions of PARP-2 and PARP-3 have beendiscovered, further expanding the utility of PARP as a target for cancerpharmacotherapy. PARP inhibitors in Phase I and Phase II clinicaltrials, used alone or in combination with known anticancer agentsinclude Olaparib (Ola), Veliparib (Veli) and Rucaparib (Ruca). Prolongedexposure to Ola and Veli leads to resistant cancer cells, primarilythrough restoration of the homologous recombination (HR) pathway,overexpression of the P-glycoprotein efflux pump or modulation of PARPexpression. Some resistant cancer cells continue to respond to platinumbased drugs, encouraging further development of PARP inhibitors forcancer treatment. Furthermore, inhibition of mitochondrial PARP has beenshown to sensitize malignant, but not non-malignant, cells toanti-cancer drugs. Thus, it is possible but has not yet been shown inhumans that the tumoricidal activity of clinically used PARP inhibitorsmay be related in part to PARP inhibition in the mitochondria. Veliparib(1,2-[(S)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide, also,ABT-888) is a promising PARP-1 inhibitor that has entered clinical phaseI/II trials for several forms of cancer including breast cancer andsolid tumor neoplasm, either as a single agent or as a combination withother chemotherapeutics.

It is therefore desirable to develop PARP inhibitors, and especiallythose with high specific activity and mitochondria-targeting ability.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant Nos.NS084604 and A1068021 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SUMMARY

If suitable PARP inhibitors could be targeted exclusively to themitochondria, it is feasible that they might prevent potentially lethalNAD⁺ depletion-associated energy failure and cell death withoutundesirable effects on genomic DNA repair; and potentially used intandem with DNA damage-targeting cancer therapies, such as radiationtherapy. The present application suggests that mitochondrially-targetedPARP inhibitors may have unique and important advantages over PARPinhibitors that are non-selectively distributed over cellularcompartments, including directly preserving NAD₊ stores withinmitochondria (a primary depot of NAD₊ within cells); preventinginitiation of MPT pore opening by a mechanism involving PARP-mediatedpost-translational modification of MPT pore components; and notimpacting facilitation of DNA repair by PARP-1 in cell nuclei.Mitochondria-targeting PARP inhibitors could have widespread clinicalefficacy in diseases where the pathophysiology includes mitochondrialdysfunction and/or energy failure, and maintaining efficient nuclear DNArepair and genomic integrity is desirable; including but not limited toischemia reperfusion injury, trauma, sub-lethal radiation injury,neurodegenerative diseases, and overwhelming infection.

Provided herein are mitochondria-targeted PARP inhibitors, such asmitochondria-targeted PARP-1 inhibitors. Based on the data presentedbelow, the disclosed mitochondria-targeted PARP-inhibitors are expectedto be therapeutically effective to treat neurodegeneration and other CNSand non-CNS conditions associated with oxidative stress, tissue damage,and cellular energy failure in a patient, as well as in combination withoncolytics. Further, the disclosed mitochondria-targeted PARP-inhibitorsare expected to be therapeutically effective to protect a patientagainst oxidative damage caused by ionizing radiation or tumoricidalagents, for example caused by chemotherapeutics or radiation therapies,a clinical need that has not yet been addressed.

Provided herein according to one aspect is a composition comprising amitochondria-targeting group covalently linked to a PARP inhibitor, or apharmaceutically-acceptable salt or ester thereof.

According to a further aspect, a method of reducing NAD₊ depletion andcell death induced by oxidative stress in a cell or a patient isprovided, comprising administering to a cell or a patient an amount of acompound comprising a mitochondria-targeting group covalently linked toa PARP inhibitor, or pharmaceutically-acceptable salt or ester thereof,effective to decrease NAD₊ depletion in mitochondria of a cell or of apatient.

According to a further aspect, a method of reducing cell death inducedby mitochondrial dysfunction and/or damage in a cell or a patient,comprising administering to a cell or a patient an amount of a compoundcomprising a mitochondria-targeting group covalently linked to a PARPinhibitor, or pharmaceutically-acceptable salt or ester thereof,effective to improve mitochondrial function and reduce mitochondrialdamage in a cell or in a patient.

According to a further aspect, a method of reducing energy failureinduced by ischemia-reperfusion in a cell or a patient, comprisingadministering to a cell or a patient an amount of a compound comprisinga mitochondria-targeting group covalently linked to a PARP inhibitor, orpharmaceutically-acceptable salt or ester thereof, effective to preventor reduce ischemia-reperfusion injury in a cell or in a patient.

According to a further aspect, a method of reducing cell death caused byexposure to ionizing radiation in a patient, comprising administering toa patient an amount of a compound comprising a mitochondria-targetinggroup covalently linked to a PARP inhibitor, orpharmaceutically-acceptable salt or ester thereof, effective to decreasemitochondrial and nuclear DNA damage and improve DNA repair in cells ofa patient.

Also provided herein according to another aspect is a method of treatinga cancer in a patient, comprising administering to a patient an amountof a first compound comprising a mitochondria-targeting group covalentlylinked to a PARP inhibitor, or pharmaceutically-acceptable salt or esterthereof, effective to sensitize malignant, but not non-malignant, cellsof a patient to anti-cancer drugs. Additional chemotherapeutic(anticancer) drugs or treatments can be administered to the patient withthe first compound.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A. PARP inhibitors in clinical use or in current trials. FIG. 1B.In FIG. 1B: 5-hydroxyisoquinolin-1(2H)-one;6-amino-1H-benzo[de]isoquinoline-1,3(2H)-dione;2-nitrophenanthridin-6(5H)-one; PD128763; E7016; and NU1025.

FIG. 2. Mitochondrially targeted 4-amino-TEMPO (XJB-5-131), lapachone(XJB-lapachone) and veliparib (XJB-veliparib). The therapeuticallyactive payload, the linker region, and the XJB mitochondrial targetingmoiety are shown in the mitochondria diagram.

FIG. 3. Synthesis of XJB-Veliparib (15).

FIG. 4. Capacity of 15 to inhibit PARP-1 enzyme. Performed intriplicate.

FIG. 5. Cytotoxicity studies in rat primary cortical neurons. LDHrelease was measured at 24 h (n=6/group).

FIGS. 6A-6C. Mitochondrial enrichment of XJB-veliparib.

FIG. 7A-7C. (FIG. 7A) OGD in rat primary cortical neurons treated with1-100 μM of veliparib or XJB-Veliparib (top panel). Bottom panel showing1-200 nM concentration range. *P<0.05 vs. naked veliparib; n=6/group.(FIG. 7B) Glutamate-glycine excitotoxicity in primary cortical neurons.Neurons were exposed to 10 μM L-glutamate and 10 μM glycine, with 1-100μM of XJB-veliparib or nontargeting veliparib for 24 h; *P<0.05 vs.naked veliparib, n=6/group. (FIG. 7C) Glutamate excitotoxicity in animmortalized hippocampal neuronal HT22 cell line. XJB-veliparib andnontargeting veliparib were both effective at inhibiting excitotoxiccell death, with XJB-veliparib slightly more effective at higher doses(*P<0.05 vs. naked veliparib; n=5/group).

FIG. 8. Mitochondrial and cytosolic NAD⁺ concentration in primarycortical neurons after OGD. Veliparib and XJB-Veliparib administered at10 nM dose before OGD. NAD₊ concentration measured 24 h after OGD. Assayperformed in duplicate.

FIG. 9. Mitochondria were labelled with anti-TOMM20 antibody (green),PADPRp were labelled with anti-PAR antibody (red in original), andnuclei were labelled with (DAPI; blue in original). Both mitochondrial(arrowheads) and nuclear (arrows) PADPRp staining were observed. PARPinhibitors were administered at 10 nM doses before OGD andimmunohistochemistry was performed 24 h after OGD.

FIG. 10. Mitochondria were labelled with anti-TOMM20 antibody (green),PADPRp were labelled with anti-PAR antibody (red in original), andnuclei were labelled with DRAQ5 (blue in original). Both mitochondrial(arrowheads) and nuclear (arrows) PADPRp staining were observed. PARPinhibitors were administered at 10 nM doses before OGD andimmunohistochemistry was performed 24 h after OGD.

FIG. 11. Graph illustrating that XJB-Veliparib reduces MEF IR-inducedcell death.

FIG. 12. Graphs showing that XJB-veliparib reduces γ-irradiation-inducedmitochondrial DNA damage.

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsalso refer to word forms, cognates and grammatical variants of thosewords or phrases.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, in reference to elements of an item, composition,apparatus, method, process, system, claim etc. are intended to beopen-ended, meaning that the item, composition, apparatus, method,process, system, claim etc. includes those elements and other elementscan be included and still fall within the scope/definition of thedescribed item, composition, apparatus, method, process, system, claimetc. As used herein, “a” or “an” means one or more. As used herein“another” may mean at least a second or more.

As used herein, the terms “patient” or “subject” refer to members of theanimal kingdom, including, but not limited to human beings.

As used herein, “alkyl” refers to straight, branched chain, or cyclichydrocarbyl groups including from 1 to about 20 carbon atoms, forexample and without limitation C₁₋₃, C₁₋₆, C₁₋₁₀ groups, for example andwithout limitation, straight, branched chain alkyl groups such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, and the like. “Substituted alkyl” refers toalkyl substituted at 1 or more, e.g., 1, 2, 3, 4, 5, or even 6positions, which substituents are attached at any available atom toproduce a stable compound, with substitution as described herein.“Optionally substituted alkyl” refers to alkyl or substituted alkyl.“Halogen,” “halide,” and “halo” refers to —F, —Cl, —Br, and/or —I.“Alkylene” and “substituted alkylene” refer to divalent alkyl anddivalent substituted alkyl, respectively, including, without limitation,ethylene (—CH₂—CH₂—). “Optionally substituted alkylene” refers toalkylene or substituted alkylene. “Cycloalkyl” refer to monocyclic,bicyclic, tricyclic, or polycyclic, 3- to 14-membered ring systems,which are either saturated, unsaturated or aromatic. The cycloalkylgroup may be attached via any atom. Cycloalkyl also contemplates fusedrings wherein the cycloalkyl is fused to an aryl or hetroaryl ring.

“Alkene or alkenyl” refers to straight, branched chain, or cyclichydrocarbyl groups including from 2 to about 20 carbon atoms, such as,without limitation C₁₋₃, C₁₋₆, C₁₋₁₀ groups having one or more, e.g., 1,2, 3, 4, or 5, carbon-to-carbon double bonds. “Alkyne or “alkynyl”refers to a straight or branched chain unsaturated hydrocarbon havingthe indicated number of carbon atoms and at least one triple bond.

The term “alkoxy” refers to an —O-alkyl group having the indicatednumber of carbon atoms. For example, a (C₁-C₆)alkoxy group includes—O-methyl (methoxy), —O— ethyl (ethoxy), —O-propyl (propoxy),—O-isopropyl (isopropoxy), —O-butyl (butoxy), —O-sec-butyl (sec-butoxy),—O-tert-butyl (tert-butoxy), —O-pentyl (pentoxy), —O-isopentyl(isopentoxy), —O-neopentyl (neopentoxy), —O-hexyl (hexyloxy),—O-isohexyl (isohexyloxy), and —O-neohexyl (neohexyloxy).

“Aryl,” alone or in combination refers to an aromatic monocyclic orbicyclic ring system such as phenyl or naphthyl. “Aryl” also includesaromatic ring systems that are optionally fused with a cycloalkyl ring.

“Heteroatom” refers to N, O, P and S. Compounds that contain N or Satoms can be optionally oxidized to the corresponding N-oxide, sulfoxideor sulfone compounds. “Hetero-substituted” refers to an organic compoundin any embodiment described herein in which one or more carbon atoms aresubstituted with N, O, P or S.

“Substituted” or “substitution” refer to replacement of a hydrogen atomof a molecule or an R-group with one or more additional R-groups such ashalogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy,mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino,alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-I-yl,piperazin-1-yl, nitro, sulfato or other R-groups.

Provided herein are mitochondrial-targeted PARP inhibitors and methodsof use for those PARP inhibitors. PARP inhibitors that targetpreferentially to the mitochondria are expected to prevent thepotentially lethal side effects of NAD⁺ depletion-associated energyfailure. As described herein, mitochondrially-targeted PARP inhibitorshave unique and important advantages over PARP inhibitors that arenon-selectively distributed over cellular compartments, includingdirectly preserving NAD₊ stores within mitochondria (a primary depot ofNAD₊ within cells); preventing initiation of MPT pore opening by amechanism involving PARP-mediated post-translational modification of MPTpore components; and not impacting facilitation of DNA repair by PARP-1in cell nuclei.

Conditions potentially treatable by the mitochondria-targeting PARPinhibitors described herein include, without limitation ischemiareperfusion injury, trauma including chronic traumatic encephalopathy,sub-lethal radiation injury, neurodegenerative diseases, andoverwhelming infection (e.g., sepsis). The data presented below supportthis. The disclosed mitochondria-targeted PARP-inhibitors are expectedto be therapeutically effective to treat neurodegeneration and other CNSand non-CNS conditions associated with oxidative stress, oxidativetissue damage, and cellular energy failure in a patient, and may besuperior to non-targeting PARP inhibitors used in combination withoncolytics. Further, the disclosed mitochondria-targeted PARP-inhibitorsare expected to be therapeutically effective to protect a patientagainst oxidative damage caused by ionizing radiation, for examplecaused by chemotherapeutics or radiation therapies, a clinical need thathas not yet been addressed.

Provided herein according to one aspect is a composition comprising amitochondria-targeting group covalently linked to a PARP inhibitor or aderivative, isostere, or pharmaceutically-acceptable salt or esterthereof.

Also provided herein according to another aspect is a method of treatinga cancer in a patient, comprising administering to a patient an amountof a first compound comprising a mitochondria-targeting group covalentlylinked to a PARP inhibitor, or a pharmaceutically-acceptable salt orester thereof, effective to treat cancer in a patient, e.g., tosensitize malignant, but not non-malignant (normal) cells in a patientto anti-cancer drugs. Additional chemotherapeutic drugs or treatmentsmay be administered to the patient with the first compound.

According to a further aspect, a method of reducing NAD₊ depletion andcell death induced by oxidative stress in a cell or a patient isprovided, comprising administering to a cell or a patient an amount of acompound comprising a mitochondria-targeting group covalently linked toa PARP inhibitor, or a pharmaceutically-acceptable salt or esterthereof, effective to decrease NAD₊ depletion in mitochondria of a cellor of a patient.

According to a further aspect, a method of reducing cell death inducedby mitochondrial dysfunction and/or damage in a cell or a patient,comprising administering to a cell or a patient an amount of a compoundcomprising a mitochondria-targeting group covalently linked to a PARPinhibitor or a pharmaceutically-acceptable salt or ester thereofeffective to improve mitochondrial function and reduce mitochondrialdamage in a cell or in a patient.

According to a further aspect, a method of reducing energy failureinduced by ischemia-reperfusion in a cell or a patient, comprisingadministering to a cell or a patient an amount of a compound comprisinga mitochondria-targeting group covalently linked to a PARP inhibitor ora pharmaceutically-acceptable salt or ester thereof effective to preventor reduce ischemia-reperfusion injury in a cell or in a patient.

According to a further aspect, a method of reducing irradiation(IR)-induced cell death and mitochondrial DNA (mtDNA) damage fromexposure to ionizing radiation in a patient, comprising administering toa patient an amount of a compound comprising a mitochondria-targetinggroup covalently linked to a PARP inhibitor, or apharmaceutically-acceptable salt or ester thereof, effective to reduceirradiation (IR)-induced cell death and mitochondrial DNA (mtDNA) damagein a patient.

A “mitochondrial targeting moiety” is a moiety (that is, a part of amolecule) that partitions specifically to mitochondria, including theirinner compartments and/or membranes. In one aspect, the mitochondriatargeting group is a peptide fragment derived from gramicidin, such as amitochondria-targeting gramicidin peptide isostere, examples of whichfollow, e.g., Leu-^(D)Phe-Pro-Val-Orn and ^(D)Phe-Pro-Val-Orn-Leuhemigramicidin fragments and isosteres thereof. Because gramicidin iscyclic, any hemigramicidin 5-mer is expected to be useful as a membraneactive peptide fragment, including Leu-^(D)Phe-Pro-Val-Orn,^(D)Phe-Pro-Val-Orn-Leu, Pro-Val-Orn-Leu-^(D)Phe,Val-Orn-Leu-^(D)Phe-Pro, and Orn-Leu-^(D)Phe-Pro-Val (from gramicidinS). Any larger or smaller fragment of gramicidin, or even largerfragments containing repeated gramicidin sequences (e.g.,Leu-^(D)Phe-Pro-Val-Orn-Leu-^(D)Phe-Pro-Val-Orn-Leu-^(D)Phe-Pro) areexpected to be useful for membrane targeting, and can readily be testedfor such activity. In one aspect, the gramicidin S-derived peptidecomprises a β-turn, which appears to confer to the peptide a highaffinity for mitochondria. Derivatives of gramicidin, or otherantibiotic fragments, include isosteres, such as (E)-alkene isosteres(see, United States Patent Publication Nos. 2007/0161573 and2007/0161544, incorporated herein by reference in their entirety, forexemplary synthesis methods).

In one aspect, an alkene peptide isostere segment of the antibioticgramicidin S (GS), the XJB mitochondria-targeting moiety describedherein, acts as an effective mitochondrial targeting vector. Thepresence of a type II′ β-turn in this pentapeptide sequence facilitatesmembrane permeability since the polar functionality of the backbone isless solvent-exposed and intramolecular hydrogen bonding is favored.

In one aspect, for example as illustrated by the Examples below, the XJBhemigramicidin S pentapeptide isostere is selected as the targeting unit(FIG. 2). The alkene peptide isostere segment in XJB is a surrogate ofthe leucyl-d-phenylalanine dipeptide in the bacterialmembrane-associated antibiotic gramicidin S (GS), and its sidechain-protected ornithylvalylproline tripeptide subunit is takendirectly from GS. The D-Phe-Pro sequence is based on the reverse turninducing sequence of GS that activates a type II′ β-turn structure thatburies several polar amide groups inside the molecule and thus mayfacilitate membrane transport. This moiety has previously been used incombination with a nitroxide payload to generate XJB-5-131, a reactiveoxygen scavenger that validated the targeting design and was found to beca. 600-fold enriched in mitochondria over the cytosol. XJB-5-131 hasshown in vivo efficacy in rodent models of Huntington's disease (HD),traumatic brain injury (TBI), ischemia-reperfusion injury, andhemorrhagic shock. In the radiation protector XJB-AMT, a nitric oxidesynthase (NOS) antagonist (AMT) is conjugated to the targeting sequence,with the goal to counteract the activation of mitochondrial NOS byionizing radiation, which can lead to inhibition of the respiratorychain, a burst of superoxide and peroxynitrite, and cellular damage.XJB-Lapachone introduces a derivative of the natural product β-lapachoneinto mitochondria and causes extensive cellular vacuolization andautophagy, as well as stimulating ROS generation in mitochondria.

In one aspect, the compound has the structure:

wherein R₁, R₂, R₅, and R₆ are independently hydrogen, hydroxyl, halo, aC₁-C₆ straight or branched-chain alkyl, or a C₁-C₆ straight orbranched-chain alkyl further comprising a phenyl group, wherein theC₁-C₆ straight or branched-chain alkyl group or the C₁-C₆ straight orbranched-chain alkyl group comprising a phenyl group is unsubstituted oris methyl-, hydroxyl- or halo-substituted, for example, and withoutlimitation, R₁, R₂, R₅, and R₆ are independently methyl-, hydroxyl- orfluoro-substituted, including: methyl, ethyl, propyl, 2-propyl, butyl,t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g., 4-hydroxybenzyl),phenyl, or hydroxyphenyl;R₄ is hydrogen, a halo, a C₁-C₆ straight or branched-chain alkyl, or aC₁-C₆ straight or branched-chain alkyl further comprising a phenyl(C₆H₅) group, wherein the C₁-C₆ straight or branched-chain alkyl groupor the C₁-C₆ straight or branched-chain alkyl group comprising a phenylgroup is unsubstituted or is methyl-, hydroxyl- or halo-substituted;R₇ is —C(O)—R₁₃, —C(O)O—R₁₃, or —P(O)—(R₁₃)₂, wherein R₁₃ is C₁-C₆straight or branched-chain alkyl or a C₁-C₆ straight or branched-chainalkyl optionally comprising one or more (C₆H₅) groups that areindependently unsubstituted, or methyl-, ethyl-, hydroxyl-,halo-substituted or fluoro-substituted, for example and withoutlimitation, R₇ is Ac (Acetyl, R=—C(O)—CH₃), Boc (R=—C(O)O-tert-butyl),Cbz (R=—C(O)O-benzyl (Bn)), or a diphenylphosphate group;R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, where R₉ is aPARP inhibitor or a derivative thereof, such as such as, olaparib,veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g., CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof, and where L is a C₁₋₅alkyl linker, optionally comprising an ester or amide linkage;R₃ is a halo, a C₁-C₆ straight or branched-chain alkyl or a C₁-C₆straight or branched-chain alkyl further comprising one or more (C₆H₅)groups that are independently unsubstituted, or methyl-, ethyl-,hydroxyl- or halo-substituted; and R₁₀, R₁₁, and R₁₂ are independently Hor halogens (See, e.g., International Patent Publication Nos. WO2010/009405 and WO 2012/112851, incorporated herein by reference intheir entirety).

In another aspect, the compound has the structure:

wherein X is

R₁, R₂, R₅, R₆, and R₁₄ are each independently hydrogen, halo, a C₁-C₆straight or branched-chain alkyl, or a C₁-C₆ straight or branched-chainalkyl further comprising a phenyl (C₆H₅) group, wherein the C₁-C₆straight or branched-chain alkyl group or the C₁-C₆ straight orbranched-chain alkyl group comprising a phenyl group is unsubstituted oris methyl-, hydroxyl- or halo-substituted;R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, where R₉ is aPARP inhibitor or a derivative thereof, such as, olaparib, veliparib,CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof, and where L is a C₁₋₅alkyl linker, optionally comprising an ester or amide linkage; andR₇ is —C(O)—R₁₃, —C(O)O—R₁₃, or —P(O)—(R₁₃)₂, wherein R₁₃ is C₁-C₆straight or branched-chain alkyl or a C₁-C₆ straight or branched-chainalkyl optionally comprising one or more (C₆H₅) groups that areindependently unsubstituted, or methyl-, ethyl-, hydroxyl-,halo-substituted or fluoro-substituted, for example and withoutlimitation, R₇ is Ac (Acetyl, R=—C(O)—CH₃), Boc (R=—C(O)O-tert-butyl),Cbz (R=—C(O)O-benzyl (Bn)), or a diphenylphosphate group.

Non-limiting examples of compounds according to (V) include:

wherein R₉ is a PARP inhibitor or a derivative thereof, such as,olaparib, veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof.

In one aspect, the compound has a structure chosen from:

wherein R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, whereR₉ is a PARP inhibitor, such as, olaparib, veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof, and where L is a C₁₋₅alkyl linker, optionally comprising an ester or amide linkage; R₁₅ andR₁₆, independently are an amine protecting group or acylated (the N isacylated). R₂₁ is H or C₁₋₃ alkyl aryl, such as methylphenyl (—CH₂-Ph).R₂₂ and R₂₃ are, independently, H, C₁₋₄alkyl or hetero-substitutedalkyl, such as a thioether, for example and without limitation, analiphatic amino acid side chain, such as

In aspects, In one aspect, R₁₅ and R₁₆ are protecting groupsindependently selected from the group consisting of:9-fluorenylmethyloxy carbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzhydryloxycarbonyl (Bhoc), benzyloxycarbonyl (Cbz),O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn), allyloxycarbonyl(alloc), trityl (Trt), I-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl(Dde), diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts),dimethoxytrityl (DMT) and monomethoxytrityl (MMT), and R₁₇ is H ormethyl. In one aspect, R₁₅ is Boc and R₁₆ is Cbz. Ph is phenyl.

In another aspect, the compound has the structure:

wherein R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, whereR₉ is a PARP inhibitor or a derivative thereof, such as, olaparib,veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof, R₁₇ is H or methyl, andwhere L is a C₁₋₅ alkyl linker, optionally comprising an ester or amidelinkage. In one aspect, in any of the compounds described herein, R₈ is:

In another aspect, the compound has the structure:

In another aspect, the compound has the structure:

wherein R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, whereR₂₀ is a PARP inhibitor or a derivative thereof, such as, olaparib,veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof, and where L is a C₁₋₅alkyl linker, optionally comprising an ester or amide linkage, and R₁₈is an amine protecting group or acylated (the N is acylated).

As used herein, unless indicated otherwise, for instance in a structure,all compounds and/or structures described herein comprise all possiblestereoisomers, individually or mixtures thereof, including anypharmaceutically-acceptable salts thereof.

A “PARP inhibitor” is an inhibitor of PARP-1, PARP-2, or PARP-3, thoughat this time, they are predominantly PARP-1 inhibitors. Non-limitingexample of PARP inhibitors, that also can be classified as PARP-1inhibitors, include olaparib, veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof (See, e.g., FIGS. 1A and1B and Curtin N.J., et al. Therapeutic applications of PARP inhibitors:anticancer therapy and beyond. Molecular aspects of medicine 2013;34:1217-56). As an example, veliparib has the structure:

Attachment (covalent linking) of the PARP inhibitor to themitochondria-targeting group (see, e.g., FIG. 2) can be achieved by anyuseful chemistry, so long as the composition substantially retains itspharmacological effect. For example, as in the examples below, veliparibis linked to a mitochondria-targeting group through its amide(carboxamide) group as in the following structure:

where R is a mitochondria-targeting moiety according to any aspectdescribed herein, and optionally comprises a linker between themitochondria-targeting group and the veliparib moiety, e.g.,

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned herein are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely the salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like. The term “addition salt”as used hereinabove also comprises the solvates which the compoundsdescribed herein are able to form. Such solvates are for examplehydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen.

Pharmaceutically acceptable counterions include chloro, bromo, iodo,trifluoroacetate and acetate. The counterion of choice can be introducedusing ion exchange resins.

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C₁₋₄ alkyl, or C₁₋₄ alkoxy) or amino); sulphonateesters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and valyl.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The term “prodrug” as used throughout this textmeans the pharmacologically acceptable derivatives such as esters,amides and phosphates, such that the resulting in vivo biotransformationproduct of the derivative is the active drug as defined in the compoundsdescribed herein. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of compounds described herein may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either by routine manipulationor in vivo, to the parent compound. The suitability and techniquesinvolved in making and using prodrugs are well known by those skilled inthe art.

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

As used herein, unless indicated otherwise, for instance in a structure,all compounds and/or structures described herein comprise all possiblestereoisomers, individually or mixtures thereof. The compound and/orstructure may be an enantiopure preparation consisting essentially of an(−) or (+) enantiomer of the compound, or may be a mixture ofenantiomers in either equal (racemic) or unequal proportions.

As used herein, a ring structure showing a bond/group that is notattached to any single carbon atom, for example and without limitation,depicted as

can be substituted at any position with one or more groups designated“R”, and, unless indicated otherwise, each instance of R on the ring canbe (independently) the same or different from other R moieties on thering. Thus, if R is H, the group contains nothing but H groups. If R is“halo”, it is a single halo (e.g., F, Cl, Br and I) group. If R is oneor more independently of halo and CN, the ring may comprise one, two,three, four, halo or CN groups, such as, for example and withoutlimitation: 2, 3, 4, or 5 chloro; 2, 3, 4, or 5 bromo; 2,3- or 3,4- or4,5- or 2,4-dichloro; 3-bromo-4-chloro; 3-bromo-4-cyano, and any otherpossible permutation of the listed groups.

Protected derivatives of the disclosed compounds also are contemplated.Many suitable protecting groups for use with the disclosed compounds arebroadly-known in the art. In general, protecting groups are removedunder conditions which will not affect the remaining portion of themolecule. These methods are well known in the art and include acidhydrolysis, hydrogenolysis and the like. One method involves the removalof an ester, such as cleavage of a phosphonate ester using Lewis acidicconditions, such as in TMS-Br mediated ester cleavage to yield the freephosphonate. A second method involves removal of a protecting group,such as removal of a benzyl group by hydrogenolysis utilizing palladiumon carbon in a suitable solvent system such as an alcohol, acetic acid,and the like or mixtures thereof. A t-butoxy-based group, includingt-butoxy carbonyl protecting groups can be removed utilizing aninorganic or organic acid, such as HCl or trifluoroacetic acid, in asuitable solvent system, such as water, dioxane and/or methylenechloride. Another exemplary protecting group, suitable for protectingamino and hydroxy functions amino is trityl. Other conventionalprotecting groups are known and suitable protecting groups can beselected by those of skill in the art in consultation with any of thelarge number of broadly-available publications. When an amine isdeprotected, the resulting salt can readily be neutralized to yield thefree amine. Similarly, when an acid moiety, such as a phosphonic acidmoiety is unveiled, the compound may be isolated as the acid compound oras a salt thereof.

According to one aspect, amine side chains are protected usingprotective groups, for example and without limitation by acylation (See,e.g., U.S. Pat. Nos. 7,528,174; 7,718,603; and 9,006,186, andInternational Patent Publication Nos. WO 2010/009405 and WO 2012/112851,incorporated herein by reference in their entirety). Protecting groupsare known in the art and include, without limitation:9-fluorenylmethyloxy carbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzhydryloxycarbonyl (Bhoc), benzyloxycarbonyl (Cbz),O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn), allyloxycarbonyl(alloc), trityl (Trt), I-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl(Dde), diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts),dimethoxytrityl (DMT) and monomethoxytrityl (MMT) groups. A protectinggroup also includes acyl groups, such as acetyl groups, for example, asdescribed.

A linker is a group that covalently attaches, in the context of thecompositions described herein, the mitochondrial-targeting moiety andthe PARP inhibitor moiety. The linker does not interfere with thepharmacological effects of the composition as a whole. Examples oflinkers include aliphatic hydrocarbons, or aliphatic hydrocarbons havingone or more aromatic groups in their structure, such as saturated orunsaturated C₁₋₁₀ hydrocarbon moieties, e.g., a linear or branchedsaturated C₁-C₁₀ alkyl. A linker, prior to incorporation into a compoundcomprises active groups, e.g., carboxyl, alkoxyl, amino, sulfhydryl,amide, etc., and a non-reactive moiety that remains once the linker isincorporated into a compound. The non-reactive moiety (such as saturatedalkyl or phenyl) does not interfere, sterically or by any other physicalor chemical attribute, such as polarity orhydrophobicity/hydrophilicity, in a negative (loss of function) capacitywith respect to the pharmacological activity of the overall compound.Linker and linking chemistry is broadly-known, and in one example, iscarbodiimide chemistry using EDC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) or DCC(N′,N′-dicyclohexyl carbodiimide) chemistry as used in the Examplesbelow (see, FIG. 3) to couple amines and carboxyl groups. Listing alllinking chemistries are broadly-known and thus are beyond the scope ofthis disclosure (see, e.g. Thermo Scientific Crosslinking TechnicalHandbook, Thermo Fisher Scientific, Inc. 2012.).

The compounds typically are administered in an amount and dosage regimento treat (a) a cancer (e.g., a malignancy), which includes, withoutlimitation, any abnormal cells that divide without control and caninvade nearby tissues, or (b) a hyperplasia, which is an increase in thenumber of cells in an organ or tissue, where the cells appear normal,and are not a cancer, but may become cancer. The compounds also areuseful in mitigating radiation damage. For example, at concentrations of5 μM or more XJB-veliparib decreased radiation damage, as described inthe examples below. The compounds also are expected to be useful intreatment of neurodegeneration includes treatment of neurodegenerativediseases, such as Parkinson's disease (PD), Alzheimer's disease (AD),Multiple Sclerosis (MS) chronic traumatic encephalopathy (CTE), andamyotrophic lateral sclerosis (ALS). The compounds may be administeredin any manner that is effective to treat, mitigate or prevent any of theabove conditions, including cancer, hyperplasia, neurodegeneration, PD,AD, MS, CTE, and ALS. Examples of delivery routes include, withoutlimitation: topical, for example, epicutaneous, inhalational, enema,ocular, otic and intranasal delivery; enteral, for example, orally, bygastric feeding tube and rectally; and parenteral, such as, intravenous,intraarterial, intramuscular, intracardiac, subcutaneous, intraosseous,intradermal, intrathecal, intraperitoneal, transdermal, iontophoretic,transmucosal, epidural and intravitreal, with oral, intravenous,intramuscular and transdermal approaches being preferred in manyinstances.

As indicated above, and in the examples, the compounds exhibitPARP-inhibiting, e.g. PARP-1-inhibiting, activities, at dosages of >0μM, e.g., >1 nM, with the upper limit being dictated by toxicity, forexample direct cellular exposure of up to 10 μM) of the compound, e.g.,as described in the examples below. Therefore, an “effective amount” ofthe compound or composition described herein is an amount effective in adosage regimen (amount of the compound and timing of delivery), toachieve a desired end-point, such as maintaining concentrations at asite of treatment within a range effective to achieve an outcome.Suitable outcomes include killing of cancer cells, improvement ormaintenance of neurological function, cellular protection includingneuroprotection, shrinking a tumor, reducing NAD⁺ depletion, ormitigating radiation damage to ionizing radiation.

The compounds may be compounded or otherwise manufactured into asuitable composition for use, such as a pharmaceutical dosage form ordrug product in which the compound is an active ingredient. Compositionsmay comprise a pharmaceutically acceptable carrier, or excipient. Anexcipient is an inactive substance used as a carrier for the activeingredients of a medication. Although “inactive,” excipients mayfacilitate and aid in increasing the delivery or bioavailability of anactive ingredient in a drug product. Non-limiting examples of usefulexcipients include: antiadherents, binders, rheology modifiers,coatings, disintegrants, emulsifiers, oils, buffers, salts, acids,bases, fillers, diluents, solvents, flavors, colorants, glidants,lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners,etc., as are available in the pharmaceutical/compounding arts.

Useful dosage forms include: intravenous, intramuscular, orintraperitoneal solutions, oral tablets or liquids, topical ointments orcreams and transdermal devices (e.g., patches). In one embodiment, thecompound is a sterile solution comprising the active ingredient (drug,or compound), and a solvent, such as water, saline, lactated Ringer'ssolution, or phosphate-buffered saline (PBS). Additional excipients,such as polyethylene glycol, emulsifiers, salts and buffers may beincluded in the solution.

In one aspect, the dosage form is a transdermal device, or “patch”. Thegeneral structure of a transdermal patch is broadly known in thepharmaceutical arts. A typical patch includes, without limitation: adelivery reservoir for containing and delivering a drug product to asubject, an occlusive backing to which the reservoir is attached on aproximal side (toward the intended subject's skin) of the backing andextending beyond, typically completely surrounding the reservoir, and anadhesive on the proximal side of the backing, surrounding the reservoir,typically completely, for adhering the patch to the skin of a patient.The reservoir typically comprises a matrix formed from a non-woven(e.g., a gauze) or a hydrogel, such as a polyvinylpyrrolidone (PVP) orpolyvinyl acetate (PVA), as are broadly known. The reservoir typicallycomprises the active ingredient absorbed into or adsorbed onto thereservoir matrix, and skin permeation enhancers. The choice ofpermeation enhancers typically depends on empirical studies. Certainformulations that may be useful as permeation enhancers include, withoutlimitation: DMSO; 95% Propylene Glycol+5% Linoleic Acid; and 50%EtOH+40% HSO+5% Propylene Glycol+5% Brij30.

Therapeutic/pharmaceutical compositions are prepared in accordance withacceptable pharmaceutical procedures.

In one aspect, the compositions as described a combined with other drugsor therapies, such as anticancer therapies, such as chemotherapeutic orradiation therapies, as are known in the art. Therefore in one aspect, amethod of treatment of a cancer is provided, comprising treating thecancer with a composition comprising a compound comprising amitochondria-targeting moiety covalently linked to a PARP inhibitor or aderivative thereof, such as, olaparib, veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof according to any aspectdescribed herein and either co-administering a second chemotherapeuticagent, or applying a radiation therapy to the patient while the compoundis present in the patient.

Chemotherapeutic agents are compounds or compositions used to treatcancer, including, for example and without limitation: abirateroneacetate, altretamine, amsacrine, anhydro vinblastine, auristatin,bafetinib, bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin, bosutinib, busulfan, cachectin, cemadotin, chlorambucil,cyclophosphamide, 3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine,docetaxol, doxetaxel, carboplatin, carmustine (BCNU), chlorambucil,cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine(DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, etoposide phosphate, 5-fluorouracil,finasteride, flutamide, hydroxyurea, hydroxyureataxanes, ifosfamide,imatinib, irinotecan, liarozole, lonidamine, lomustine (CCNU), MDV3100,mechlorethamine (nitrogen mustard), melphalan, mitoxantrone, mivobulinisethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,nilotinib, nilutamide, onapristone, oxaliplatin, paclitaxel, ponatinib,prednimustine, procarbazine, RPRI 09881, stramustine phosphate,tamoxifen, tasonermin, taxol, teniposide, topotecan, tretinoin,vinblastine, vincristine, vindesine sulfate, and vinflunine, andpharmaceutically acceptable salts thereof.

Example 1

Synthesis of XJB-veliparib: The chemical synthesis of themitochondria-targeting XJB-veliparib hybrid molecule is shown in FIG. 3.The XJB pentapeptide isostere was chosen as the targeting unit (FIG. 2).For the synthesis of mitochondrially targeted veliparib, XJB-Veliparib,N-Cbc-L-proline methyl ester (7) was treated with NaHMDS and Melfollowed by hydrolysis to give the acid 9 in 98% yield over 2 steps(Scheme 1). One-pot EDCl coupling to methyl 2,3-diaminobenzoate (10) andacid-catalyzed cyclization provided the benzimidazole 11 in good overallyield. Hydrolysis of the methyl ester followed by acylation with theN-Boc-1,3-diaminopropane spacer group afforded 12. Removal of theCbz-group via hydrogenation followed by Boc-deprotection provided 13,which was coupled to the Boc-Leu-D-Phe-Pro-Val-Orn(Cbz)-OH targetingsequence 14 to afford the desired XJB-Veliparib conjugate 15.

In further detail, all moisture- and air-sensitive reactions wereperformed in oven dried glassware under a positive pressure of argon.All reagents and solvents were used as received unless otherwisespecified. THE and Et₂O were distilled over sodium/benzophenone ketyl;CH₂Cl₂ was distilled over CaH₂, MeCN and DMF were dried over molecularsieves. Reactions were monitored by TLC analysis (pre-coated silica gel60 F₂₅₄ plates, 250 μm layer thickness) and visualization wasaccomplished with a 254/280 nm UV light and/or by staining with KMnO₄solution (1.5 g KMnO₄ and 1.5 g K₂CO₃ in 100 mL of a 0.1% NaOHsolution), a ninhydrin solution (2 g ninhydrin in 100 mL EtOH), a PMAsolution (5 g phosphomolybdic acid in 100 mL EtOH), or a p-anisaldehydesolution (2.5 mL p-anisaldehyde, 2 mL AcOH and 3.5 mL conc. H₂SO₄ in 100mL EtOH). Flash chromatography was performed on silica gel (40-63 μm).Melting points were determined on a Mel-Temp II capillary melting pointapparatus fitted with a Fluke 51 II digital thermometer. Infraredspectra were recorded on an ATR spectrometer. NMR spectra were recordedon 300, 400, 500 or 700 MHz instruments. Chemical shifts were reportedin parts per million (ppm) and referenced to residual solvent. ¹H NMRspectra are tabulated as follows: chemical shift, multiplicity(br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet),coupling constant(s), number of protons. ¹³C NMR spectra were obtainedusing a proton-decoupled pulse sequence and are tabulated by observedpeak. LC-MS analyses were performed on a Shimadzu UFLC instrumentequipped with an Applied Biosystem MDS SCIEX API 2000 mass spectrometer(ESI), under the following conditions: column: Varian Polaris C18-A(100×4.6 mm, 5 μm) equilibrated at 40° C.; buffer A: 0.1% aqueous AcOH,buffer B: 0.1% AcOH in MeCN; 30 min gradient: 5% buffer B in buffer Afor 1 min, then 5 to 95% buffer B in buffer A over 13 min, then 95%buffer B in buffer A for 4 min, then 95-5% buffer B in buffer A over 7min, then 5% buffer B in buffer A for 5 min; flow rate: 0.2 mL/min;detection: TIC and/or UV A=254/280 nm.

1-benzyl 2-methyl (S)-2-methylpyrrolidine-1,2-dicarboxylate (8). To a−78° C. solution of N—Z-L-proline methyl ester 7 (0.43 mL, 1.9 mmol) andiodomethane (0.24 mL, 3.8 mmol) in THE (3.5 mL) was added NaHMDS (1 M inTHF, 3.8 mL, 3.8 mmol) dropwise. The resulting mixture was warmed to−20° C. and stirred at this temperature for 3 h. The mixture wasquenched with H₂O, acidified with 2N HCl and extracted with EtOAc (3×).The combined organic layers were washed with brine (1×), dried overMgSO₄, filtered and concentrated to dryness. The residue was purified bychromatography on SiO₂ (30% EtOAc/hexanes) to give 8 as a pale yellowoil (0.52 g, 99%). Spectral data are in accordance with literaturevalues (Penning, T. D., et al., Discovery of the Poly(ADP-ribose)Polymerase (PARP) Inhibitor2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide (ABT-888)for the Treatment of Cancer. J. Med. Chem. 2009, 52, 514-523). ¹H NMR(300 MHz, CDCl₃) δ 1.54 (s, 1.5H), 1.61 (s, 1.5H), 1.86-1.99 (m, 3H),2.13-2.24 (m, 1H), 3.46 (s, 1.5H), 3.56-3.69 (m, 2H), 3.71 (s, 1.5H),4.99-5.23 (m, 2H), 7.24-7.40 (m, 5H) (as a mixture of two rotamers).

(S)-1-((benzyloxy)carbonyl)-2-methylpyrrolidine-2-carboxylic acid (9).Methyl ester 8 (1.53 g, 5.52 mmol) was dissolved in THE (10.5 mL) andH₂O (5.4 mL) and treated with a solution of LiOH (265 mg, 11.0 mmol) inH₂O (5.4 mL). MeCOH (1.5 mL) was then added and the resulting homogenoussolution heated to 60° C. overnight. The organic solvents were removedand the aq. layer acidified to pH 2 using 2N HCl and extracted withEtOAc (3×). The combined organic layers were washed with water (1×),dried (MgSO₄) filtered and concentrated to dryness to give 9 as a whitesolid (1.42 g, 98%): Spectral data are in accordance with literaturevalues (Penning, T. D., et al., J. Med. Chem. 2009, 52, 514-523). ¹H NMR(500 MHz, DMSO-d₆) δ 1.44 (s, 1.5H), 1.45 (s, 1.5H), 1.80-1.96 (m, 3H),2.02-2.19 (m, 1H), 3.43-3.53 (m, 2H), 4.94-5.11 (m, 2H), 7.24-7.41 (m,5H), 12.51 (br s, 1H) (as a mixture of two rotamers).

Methyl(S)-2-(1-((benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-benzo-[d]imidazole-4-carboxylate(11). To a solution of 9 (200 mg, 0.760 mmol) and Methyldiaminobenzoate10 (189 mg, 1.14 mmol) in CH₂Cl₂ (15 mL) was added DIPEA (0.13 mL, 0.76mmol) followed by addition of EDCl (218 mg, 1.14 mmol), HOAt (155 mg,1.14 mmol) and DMAP (9.3 mg, 0.076 mmol) and the resulting solution wasstirred at rt overnight. The reaction mixture was quenched with sat. aq.NH4Cl solution and extracted with portions of CH₂Cl₂ (2×). The combinedorganic layers were washed with brine (1×), dried (MgSO₄), filtered andevaporated to give the coupled product as a brown oil. The crude residuewas redissolved in AcOH (5 mL) and heated at reflux for 2 h. The solventwas evaporated, the crude poured onto sat. aq. NaHCO₃ and extracted withEtOAc (3×). The combined organic layers were washed with brine (1×),dried (MgSO₄) and the solvent evaporated. The crude residue was purifiedvia chromatography on SiO₂ (30-90% EtOAc/hexanes) to give 11 as a paleyellow oil (198 mg, 65% over 2 steps): HRMS (ESI⁺) m/z calcd forC₂₂H₂₄N₃O₄ [M+H] 394.1761, found 394.1758.

(S)-2-(1-((benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxylicacid. Methyl ester 11 (280 mg, 0.712 mmol) was dissolved in THF/H₂O(14:1, 21 mL) and cooled to −5° C. A 40% n-Bu₄NOH (aq.) solution (4.6mL, 7.1 mmol) was added slowly and the reaction mixture stirred for 30min at −5° C. and at rt over night. The solution was acidified with aq.AcOH and extracted with portions of EtOAc (3×). The combined organiclayers were washed with brine, dried (MgSO₄), filtered and the solventremoved under reduced pressure. The crude was purified via columnchromatography on SiO₂ (0-10% MeOH/CH₂Cl₂) to give(S)-2-(1-((benzyloxy)carbonyl)-2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxylicacid as a pale-yellow foam (215 mg, 80%): ¹H NMR (400 MHz, DMSO-d₆) δ7.86 (br s, 1H), 7.78 (d, J=7.5 Hz, 1H), 7.41-7.36 (m, 4H), 7.33-7.30(m, 1H), 7.30-7.25 (m, 1H), 7.06 (t, J=7.4 Hz, 0.5H), 6.91 (t, J=7.0 Hz,1H), 6.70 (br s, 1H), 5.15-5.09 (m, 0.5H), 5.07-5.04 (m, 1H), 3.87-3.54(m, 3H), 2.76 (br s, 1H), 1.92 (s, 3H), 1.87 (br s, 2H), 1.86 (br s,1H); ¹³C-NMR (700 MHz, DMSO-d₆) δ 167.5, 160.5, 159.8, 154.4, 154.1,137.4, 136.8, 128.9, 128.2, 127.9, 127.7, 127.1, 124.4, 124.3, 121.7,66.3, 66.2, 62.9, 55.4, 48.8, 48.3, 42.7, 40.5, 24.4, 24.2, 22.9, 22.4;IR (ATR, neat) 1682.8, 1407.6, 1351.5, 1255.8, 746.4 cm⁻¹; Mp102.3-104.6° C.; HRMS (ESI⁺) m/z calcd for C₂₁H₂₂N₃O₄ [M+H] 380.1605,found 380.1610.

benzyl(S)-2-(4-((3-((tert-butoxycarbonyl)amino)propyl)carbamoyl)-1H-benzo-[d]imidazol-2-yl)-2-methylpyrrolidine-1-carboxylate(12). To a solution of N-Boc-1,3-propanediamine (0.28 mL, 1.6 mmol) and11a (342 mg, 1.05 mmol) in CH₂Cl₂ (12 mL) at 0° C. was added DIPEA (0.46mL, 2.6 mmol) followed by the dropwise addition of T₃P (50 wt % inEtOAc, 0.92 mL, 1.6 mmol). The resulting mixture was warmed and stirredat rt overnight, washed with 5% aq. Na₂CO₃ and brine, dried (MgSO₄),filtered and evaporated. The crude was purified via chromatography onSiO₂ (100% CH₂Cl₂ to 10% MeOH/CH₂Cl₂) to give 12 as a pale-yellow foam(503 mg, 89%): ¹H NMR (600 MHz, DMSO-d₆) δ 12.76 (br s, 0.5H), 12.69 (brs, 0.5H), 9.93 (br s, 1H), 7.86-7.80 (m, 1H), 7.64 (d, J=7.7 Hz, 0.5H),7.60 (d, J=7.7 Hz, 0.5H), 7.39-7.34 (m, 2H), 7.33-7.27 (m, 2H),6.94-6.81 (m, 2H), 6.73-6.65 (m, 1H), 5.09-4.97 (m, 1H), 4.94-4.79 (m,1H), 3.86-3.77 (m, 1H), 3.70-3.60 (m, 1H), 3.47-3.37 (m, 2H), 3.11-3.00(m, 2H), 2.28-2.10 (m, 2H), 2.02-1.94 (m, 2H), 1.91 (br s, 1.5H), 1.89(br s, 1.5H), 1.73-1.65 (m, 3H), 1.38 (s, 9H); ¹³C-NMR (600 MHz,DMSO-d₆) δ 165.1, 160.4, 160.2, 156.1, 154.1, 153.7, 140.7, 140.6,137.5, 136.7, 135.3, 135.2, 128.8, 128.2, 127.8, 127.7, 127.0, 122.5,122.4, 122.3, 122.2, 122.1, 115.2, 115.1, 77.9, 66.2, 62.6, 62.2, 55.4,49.0, 48.2, 43.4, 42.1, 38.2, 36.9, 30.3, 30.3, 28.7, 24.4, 23.3, 23.0,22.5 (as a mixture of rotamers); Mp 112.7-116.2° C.; HRMS (ESI⁺) m/zcalcd for C₂₉H₃₈N₅O₅ [M+H] 536.2867, found 536.2867.

tert-butyl(S)-(3-(2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carbox-amido)propyl)carbamate.A solution of 12 (500 mg, 0.933 mmol) and 10% Pd/C (99 mg, 0.093 mmol)in MeCOH (5 mL) was placed in the Anton Paar hydrogenator and thereaction mixture was flushed with argon and purged with hydrogen (3×).The pressure was set to 6 bar and the mixture stirred at this pressureat rt overnight. The solution was filtered through a plug of CELITE© andwashed with portions of CH₂Cl₂. The solvent was evaporated and the crudematerial was purified via chromatography on SiO₂ (100% CH₂Cl₂ to 10%MeOH/CH₂Cl₂) to give tert-butyl(S)-(3-(2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamido)propyl)carbamateas a white foam (375 mg, 85%). ¹H NMR (600 MHz, DMSO-d₆) δ 12.50 (br s,2H), 9.94 (br s, 1H), 7.78 (d, J=7.5 Hz, 1H), 7.62 (d, J=7.5 Hz, 1H),7.25 (t, J=7.7 Hz, 1H), 6.92-6.87 (m, 1H), 3.45-3.38 (m, 2H), 3.12-3.04(m, 3H), 2.93-2.86 (m, 1H), 2.45-2.38 (m, 1H), 1.89-1.80 (m, 2H),1.72-1.64 (m, 3H), 1.59 (br s, 3H), 1.38 (s, 9H); ¹³C-NMR (600 MHz,DMSO-d₆) δ 165.3, 156.2, 122.1, 121.8, 79.7, 79.4, 79.2, 77.9, 62.6,60.2, 55.4, 49.1, 46.4, 38.1, 36.8, 30.4, 28.7, 27.6, 25.7, 21.3, 14.6;IR (ATR, neat) 3269, 2973, 1694, 1645, 1612, 1523, 1406, 1365, 1245,1166, 1046, 988, 758 cm⁻¹; Mp 79.8-83.2° C.; HRMS (ESI⁺) m/z calcd forC₂₁H₃₂N₅O₃ [M+H] 402.2500, found 402.2499.

(S)—N-(3-aminopropyl)-2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamidehydrochloride (13). To a solution of tert-Butyl(S)-(3-(2-(2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazole-4-carboxamido)propyl)carbamate(32.8 mg, 0.0817 mmol) in CH₂Cl₂ (1 mL) was added 4M HCl in dioxane (0.2mL, 0.8 mmol). The resulting mixture was stirred at rt for 2 h. Thedesired product was filtered off, washed with portions of hexanes andthe resulting white solid (27.1 mg, 98%) was used in the next stepwithout further purification. HRMS (ESI⁺) m/z calcd for C₁₆H₂₄N₅O [M+H]302.1975, found 302.1974.

tert-Butyl((4S,7S,E)-7-benzyl-2-methyl-8-((S)-2-(((S)-3-methyl-1-(((S)-15-(2-((S)-2-methylpyrrolidin-2-yl)-1H-benzo[d]imidazol-4-yl)-3,9,15-trioxo-1-phenyl-2-oxa-4,10,14-triazapentadecan-8-yl)amino)-1-oxobutan-2-yl)carbamoyl)pyrrolidin-I-yl)-8-oxooct-5-en-4-yl)carbamate(“XJB-Veliparib”, 15). To a solution of XJB-acid 14 (36.9 mg, 0.0458mmol) and 13 (23.2 mg, 0.0687 mmol) in DMF (0.92 mL, 0.05M) at 0° C. wasadded DIPEA (40 μL, 0.23 mmol) followed by the dropwise addition of T₃P(50% in DMF, 35 μL, 0.060 mmol). The reaction mixture was stirred at 0°C. for 1 h, after which it was quenched with aq. NH4Cl, and extractedwith CH₂Cl₂ (3×). The combined organic layers were washed with 5% aq.LiCl solution (1×), dried (MgSO₄), filtered and evaporated to give thecrude product as a pale yellow oil. The residue was purified viachromatography on SiO₂ (100% CH₂Cl₂ to 15% MeOH/CH₂Cl₂) to afford 15 asa white solid (18.5 mg, 37%). ¹H NMR (400 MHz, DMSO-d₆, 100° C.) δ 9.32(br s, 1H), 7.88 (d, J=7.44 Hz, 1H), 7.72 (d, J=7.96 Hz, 1H), 7.69-7.59(m, 1H), 7.47-7.38 (m, 1H), 7.37-7.31 (m, 5H), 7.30-7.26 (m, 1H),7.25-7.12 (m, 5H), 6.79 (br s, 1H), 6.12 (d, J=7.72 Hz, 1H), 5.52-5.42(m, 2H), 5.02 (s, 2H), 4.44-4.37 (m, 1H), 4.30-4.23 (m, 1H), 4.18-4.09(m, 1H), 3.93-3.84 (m, 1H), 3.53-3.35 (m, 6H), 3.29-3.21 (m, 3H),3.10-3.03 (m, 4H), 2.72-2.00 (m, 3H), 1.98-1.88 (m, 2H), 1.84 (br s,3H), 1.80-1.71 (m, 5H), 1.66-1.57 (m, 1H), 1.55-1.43 (m, 3H), 1.38 (s,9H), 1.30-1.25 (m, 4H), 0.90-0.78 (m, 12H); ¹³C NMR (700 MHz, DMSO-d₆) δ172.7, 172.5, 172.2, 171.9, 171.8, 171.7, 171.4, 171.3, 171.2, 164.8,156.6, 155.3, 155.2, 139.7, 139.5, 137.7, 135.0, 134.6, 129.6, 128.8,128.4, 128.3, 128.2, 127.8, 126.4, 126.2, 122.8, 78.0, 77.8, 65.6, 64.9,64.6, 59.7, 59.6, 59.3, 58.2, 53.1, 53.0, 50.7, 50.5, 49.3, 48.8, 47.2,47.1, 45.5, 38.9, 38.2, 36.5, 36.4, 35.2, 32.4, 30.9, 30.7, 30.2, 29.8,28.7, 26.5, 25.0, 24.7, 24.4, 24.3, 23.8, 23.0, 22.8; Mp 147.2-152.6°C.; IR (ATR, CH₂Cl₂) 3291.7, 2955.8, 1647.1, 1528.8, 1439.1, 1365.7,1248.0, 1167.2, 1028.3, 758.2, 698.1 cm⁻¹; HRMS (ESI) m/z calcd forC₆₀H₈₅N₁₀O₉ 1089.6496, found 1089.6495.

PARP1 Activity: The capacity to inhibit PARP1 was determined using acommercial assay (Trevigen, Gaithersburg, Md.) as per manufacturer'sdirection. Various concentrations of XJB-Veliparib or naked veliparibwere added to histone-coated wells containing active PARP1 enzyme andNAD⁺ in surplus.

Quantification of XJB-veliparib: The mitochondrial fraction (50 μL) ornuclear fraction (50 μL), treated with XJB-veliparib or veliparib (100nM), was added to a 5:1 ratio of CH₂Cl₂:MeOH (950 μL) and vortexed (30sec). Water (150 μL) was added and the solution was vortexed (15 sec)and set aside to equilibrate at room temperature (30 min). The resultingsuspension was placed in an Eppendorf Centrifuge 5702 (4400 rpm, 20° C.)for 12 min. The organic layer was extracted and filtered through a 0.45μm filter for analysis.

XJB-Veliparib and veliparib content was quantified on a ThermoScientific Exactive Orbitrap LC-MS (ESI positive ion mode) coupled to aThermo Scientific Accela HPLC system using a 3.5 μM Water XTerra C18column (2.1×50 mm; 20 min gradient elution with MeCN/H₂O containing 0.1%formic acid at a flow rate of 500 μL/min from 5:95 at 0-1.0 min to 95:5at 12.0 min, back to 5:95 from 16.0 to 16.1 min). Calibration curves forXJB-Veliparib and veliparib were run in duplicate from 102 nM to 5.7 nM.Samples (10 μL) were injected in triplicate and Thermo Xcalibur softwarewas used to determine the concentration of XJB-Veliparib and veliparibin mitochondrial and nuclear fractions (n=3). The concentration wasreported as pM concentration of XJB-Veliparib per 10 μg of protein withcorresponding standard deviation values.

Cell Cultures: Primary cortical neuron-enriched cultures were preparedfrom 16-17 day old Sprague-Dawley rat embryos. Dissociated cellsuspensions were filtered through a 70 μm nylon cell strainer and seededin 96-well plates (5×10⁴ cells/well) or on poly-D-lysine coated glasscoverslips, and maintained in Neurobasal medium with B27 supplements(Life Technologies, Carlsbad, Calif.). Experiments were performed 12days in vitro (DIV).

HT22 cells were cultured at 37° C. in Dulbecco's modified Eagle's medium(DMEM) (Invitrogen Inc., Carlsbad, Calif.) supplemented with 10% fetalbovine serum (FBS) (Thermo Fisher Scientific, San Jose, Calif.) and 1%penicillin-streptomycin (ATCC, Manassas, Va.) in an atmospherecontaining 5% CO₂. Cells were cultured for 24 to 48 hr before use.

Oxygen-glucose Deprivation: To model ischemia-reperfusion in vitro,culture medium was replaced with a pre-equilibrated low glucose (0.5 mM)medium. Neurons were transferred into a sealed hypoxic chamber (CoyLaboratory Products Inc., Grass Lake, Mich.) set to an atmosphere of 95%N₂ with 5% CO₂ at 37° C. for 2 h. After OGD neurons were removed fromthe chamber and returned to the incubator.

Excitotoxicity: To model excitotoxicity in primary cortical neurons,cells were exposed to 10 μM L-glutamate with 10 μM glycine. Neuronalcells from HT22 cell line were exposed to 5 mM L-glutamate.

Assessment of Cell Death: Cell death was quantified by measuring lactatedehydrogenase (LDH) released into supernatant using a colorometricassay. LDH values were normalized to 100% cell death caused by 0.5%Triton X-100 exposure. Data are reported as the percentage of dead cellsrelative to total cells and presented as mean±standard deviation (SD).

Immunocytochemistry and Immunofluorescent Microscopy: Neurons grown onpoly-D-lysine coated glass coverslips were fixed in 2% paraformaldehydeand permeabilized with TritonX-100. Coverslips were then incubated in a1:200 dilution of mouse monoclonal antibody against PAR (SA216, ENzoLife Sciences, Inc., Farmingdale, N.Y.) and an antibody against TOMM20(Abcam, Cambridge, Mass.) followed by incubation in the appropriatesecondary antibodies. Cell nuclei were labelled with 4′,6diamidino-2-phenylindole (DAPI) for standard confocal microscopy orDRAQ5 (both from ThermoFisher Scientific, Waltham, Mass.) for STEDmicroscopy. Images were collected using an Olympus Fluoview 1000confocal microscope (Olympus Corporation of the Americas, Center Valley,Pa.).

For STED imaging a Leica TCS SP8 super resolution STED microscope with apulsed white light laser and AOBS detection system was used (LeicaMicrosystems, Wetzlar, Germany). Images were collected using the 775 nmSTED laser line with 30% 3D STED using the Leica STED WHITE oilobjective lens (HC PL APO 100×/1.40 OIL) with a 200 Hz scan speed and 2×line averaging. Pixel size was set to 45 nm/pixel, step size was set to160 μm, and pinhole was set at 132.8 μm (0.875 AU). TOMM20 wasvisualized using Alexa Fluor 555, exciting at 553 nm and detectingbetween 558-599 nm and temporally gate between 0.83-4.33 nm. PAR wasvisualized with Alexa Fluor 594, exciting at 598 nm and detectingbetween 603-666 nm and temporally gated between 0.3-6.0 nsec. DRAQ5(ThermoFisher Scientific, Waltham, Mass.) was excited at 662 anddetected between 667 to 780 nm and temporally gated between 0.3-6.0nsec. Channels were collected between stacks, sequentially.

PARP Inhibition ex vivo, Cytotoxicity, and Mitochondrial EnrichmentStudies: To determine whether the linkage to the mitochondria-targetingmoiety on 15 affected PARP1 inhibition, a control assay with activePARP1 enzyme was used. PARP1 inhibition was similar between untargetedveliparib and the XJB-veliparib conjugate 15 (FIG. 4; experimentsperformed in triplicate), and is consistent with the reported K_(i) ofveliparib of 5.2 nmol/L.

To investigate the biological properties and potential cytotoxicity ofXJB-veliparib, rat primary cortical neurons were exposed to varyingconcentrations of untargeted veliparib and XJB-veliparib. Cytotoxicitywas assessed by LDH release from dying neurons at 24 h. While bothveliparib and XJB-veliparib showed a dose-dependent cytotoxicityprofile, XJB-veliparib was significantly less toxic compared withunconjugated veliparib (FIG. 5; n=6/group; *P<0.05). Neurotoxicitydefined as >10% cell death was seen with veliparib at 1 μMconcentration, vs. a 10 μM concentration required for XJB-veliparib.Significant cytotoxicity has previously been reported when leukemiacells are exposed to micromolar concentrations of veliparib. Sincecytotoxicity produced by PARP inhibitors in clinical use, includingveliparib, may be related to genomic instability, mitochondria-targetingPARP inhibitors may have a therapeutic advantage where prevention ofcell death is desired.

To verify mitochondrial enrichment, rat primary cortical neurons wereexposed to 100 nM XJB-veliparib for 24 h. Mitochondria- andnuclear-enriched subcellular fractions were obtained and verified bywestern blot using antibodies against the cytochrome oxidase subunit Vα(COX Vα) and histone H3, respectively (FIG. 6A). Inmitochondria-enriched fractions, the LCMS analysis of XJB-veliparibindicated a concentration of 116±55 pmol/10 μg protein compared with29±27 pmol/10 μg protein in nuclear-enriched fractions (FIG. 6B;mean±standard deviation [SD]; n=3 independent experiments). In addition,mitochondria-enriched fractions spiked with active PARP1 enzyme showedgreater PARP1 inhibitory capacity compared to unconjugated veliparib(FIG. 6C; performed in triplicate).

XJB-veliparib Neuroprotection Studies: To determine whethermitochondria-targeting XJB-veliparib can promote neuronal survival inischemia-like conditions in vitro, primary cortical neurons at 12 DIVwere subjected to OGD. Cultured neurons were exposed to a hypoxic andglucose-depleted environment for 2 h, followed by normal cultureconditions for 24 h to mimic ischemia/reperfusion injury. As shown inFIG. 7A, treatment with XJB-veliparib significantly attenuatedOGD-induced cell death at low nanomolar concentrations. Specifically, 10nM XJB-veliparib reduced cell death by 67% (LDH release 12.4±3.1% vs.38.0±2.5%, 10 nM XJB-veliparib vs. vehicle; mean±SD; P<0.05). Treatmentwith concentrations >100 nM of either veliparib or XJB-veliparibconjugate provided no additional protection. Both XJB-veliparib andnon-targeting veliparib appear more protective against OGD compared withother published PARP1 inhibitors, where protection is observed inmicromolar ranges. In vivo, PARP1 inhibition is highly effective atreducing neuronal death caused by ischemia-reperfusion injury.

To determine whether XJB-veliparib was also effective in attenuatingexcitotoxic cell death in vitro, primary cortical neurons were exposedto 10 μM L-glutamate and 10 μM glycine with varying concentrations ofXJB-veliparib or naked veliparib for 24 h. PARP inhibition reduced celldeath after glutamate/glycine exposure in a dose-dependent manner (FIG.7B). Differing from in vitro ischemia/reperfusion, non-targetingveliparib was more potent than XJB-veliparib in reducing LDH release.This is consistent with previous studies showing an important role fornuclear PARP1 activation in inhibiting excitotoxic neuronal death invitro (Zhang, J., et al., Nitric oxide activation of poly(ADP-ribose)synthetase in neurotoxicity. Science 1994, 263 (5147), 687-9). In vivo,PARP1 inhibition is effective at reducing N-methyl-D-aspartate (NMDA)but not non-NMDA excitotoxicity (Mandir, A. S., et al., NMDA but notnon-NMDA excitotoxicity is mediated by Poly(ADP-ribose) polymerase. JNeurosci 2000, 20 (21), 8005-11).

In addition to rat primary cortical neuron cultures, the effect ofveliparib and XJB-veliparib was determined in a stable mouse hippocampalneuronal cell line HT22, in which ferroptotic cell death is induced byhigh concentrations of glutamate. HT22 cells grown to confluence wereexposed to 5 mM glutamate and various concentrations of veliparib orXJB-veliparib with LDH release measured at 24 h (FIG. 7C). Bothveliparib and XJB-veliparib inhibit glutamate-induced ferroptosis inHT22 cells, with XJB-veliparib slightly more effective than veliparib athigher concentrations (10 μM).

In order to investigate the cellular site of action of both velipariband XJB-veliparib, the NAD⁺ concentrations in mitochondria- andcytosol-enriched fractions were measured after OGD. Both XJB-velipariband non-targeting veliparib preserve mitochondrial NAD⁺ stores (FIG. 8).However, while OGD led to increased cytosolic NAD⁺ levels, it was foundthat a 10 nM dose of XJB-veliparib prevented efflux of NAD⁺ from themitochondria to the cytosol 24 h after OGD.

Cellular Localization of Poly(ADP-ribose) Polymers: The cellularlocalization of poly(ADP-ribose) (PAR) polymers, a footprint of PARPactivation, was examined in primary cortical neurons after OGD usingimmunohistochemistry and confocal microscopy. Primary rat corticalneurons were treated with 10 nM XJB-veliparib, 10 nM veliparib, orvehicle and then exposed to OGD for 2 h. As shown in FIG. 9, one hourafter OGD in vehicle treated neurons, PARP activation, assessed byimmunofluorescence staining with anti-PAR antibody, was increased inmitochondria (labelled with translocase of outer mitochondrial membrane20 [TOMM20]) and nuclei (labelled with 4,6-diamidino-2-phenylindole[DAPI]) compared with control neurons (no ischemia; performed intriplicate). After treatment with either veliparib or XJB-veliparib, areduction of PAR immunofluorescence was observed. The PAR data obtainedwere consistent with PARP activation in mitochondria after OGD,inhibited by the XJB-veliparib conjugate and the unconjugated veliparib.The relative increase in PAR immunoreactivity observed in mitochondriavs. nuclei in neurons after OGD may be explainable by more effective PARmetabolism by poly(ADP-ribose) glycohydrolase in cell nuclei vs.ADP-ribosylhydrolase 3 in mitochondria.

To evaluate the impact of XJB-veliparib on mitochondrial structure afterOGD, we used stimulation emission depletion (STED) microscopy andimmunohistochemistry. Primary rat cortical neurons were treated with 10nM XJB-veliparib or vehicle and then exposed to OGD for 2 h. Vehicletreated neurons showed increased PARP activation at 1 h as determined byPAR immunohistochemistry, and swollen, circular mitochondria consistentwith fission compared with control (no ischemia) neurons (FIG. 10;performed in triplicate). In contrast, PAR immunoreactivity was reducedin neurons treated with XJB-veliparib (vs. vehicle) and mitochondrialarchitecture appeared partially preserved.

Example 2—XJB-Veliparib Mitigates the Effects of Radiation Exposure

XJB-veliparib reduces IR-induced cell death and mitochondrial DNA(mtDNA) damage in mouse embryo fibroblasts (MEF), suggesting thatXJB-veliparib may be an effective mitigator of radiation toxicity. FIG.11 shows that XJB-Veliparib reduces irradiation death in MEFs. MEF cellswere treated with 10, 20 μM XJB-veliparib for 30 min prior to 10 Gy IR(309R/min, Cs137 source), media was changed to drugless media 4 hpost-IR. Survival was assessed at 48 h by Annexin V-FTIC/PI flowcytometry. MEFs treated with XJB-veliparib had increased survival vs.vehicle or veliparib (90.1%±0.3 vs. 68.4%±2.2 or 57.1%±0.1.9,respectively). Mean±SD, *p<0.05, ****p<0.0001.

FIG. 12 shows that XJB-veliparib reduces γ-irradiation-inducedmitochondrial DNA damage in MEFs. PARylation of mtDNA repair enzymes,polymerase-γ and exo/endonuclease G, by mt-PARP1 is thought interferewith normal mtDNA repair. MEFs were treated with 20 μM XJB-veliparib orunconjugated veliparib for 30 minutes prior to 10 Gy IR (309R/min, Cs137source), media was changed to drugless media 4 h post-IR. Cells wereharvested and DNA extracted 4 and 24 h post-IR. Specific inhibition ofmt-PARP1 by XJB-veliparib reduced the number of PCR-detectable mtDNAlesions (increased qPCR amplification efficiency) following γ-IRcompared to vehicle-treated (0.30±0.03 vs. 0.48±0.03, respectively).Veliparib had no effect compared to vehicle irradiated cells. ANOVA,*p<0.05, **p<0.01.

The following numbered clauses describe non-limiting various aspects ofthe present invention.

1. A compound comprising a mitochondria-targeting gramicidin S peptideisostere moiety covalently linked to a PARP inhibitor, or apharmaceutically-acceptable salt or ester thereof.2. The compound of clause 1, having the structure:

wherein R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, whereR₉ is a PARP inhibitor, such as, olaparib, veliparib, CEP-8983(II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, PD128763, andanalogues, isosteres, and derivatives thereof, and where L is a C₁₋₅alkyl linker, optionally comprising an ester or amide linkage;R₁₅ and R₁₆, independently are an amine protecting group or acylated;R₂₁ is H or C₁₋₃ alkylaryl, such as methylphenyl (—CH₂-Ph);R₂₂ and R₂₃ are, independently, H, C₁₋₄alkyl or hetero-substitutedalkyl, such as a thioether, for example and without limitation, analiphatic amino acid side chain, such as

or a pharmaceutically acceptable salt or ester thereof.3. The compound of clause 2, having the structure:

or a pharmaceutically acceptable salt or ester thereof.4. The compound of clause 2, having the structure:

or a pharmaceutically acceptable salt or ester thereof.5. The compound of any one of clauses 2-4, wherein R₂₂ and R₂₃ are,independently, aliphatic amino acid side chains, such as

6. The compound of clause 2, having the structure:

or a pharmaceutically acceptable salt or ester thereof.7. The compound of any one of clauses 2-6, wherein R₁₅ is Boc and R₁₆,when present, is Cbz.8. The compound of clause 2, having the structure:

pharmaceutically-acceptable salt or ester thereof.9. The compound of any one of clauses 2-8, wherein R₈ is:

or a pharmaceutically-acceptable salt or ester thereof.10. The compound of clause 1, having a structure:

-   -   wherein R₁, R₂, R₅, and R₆ are independently hydrogen, hydroxyl,        halo, a C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆        straight or branched-chain alkyl further comprising a phenyl        (C₆H₅) group, wherein the C₁-C₆ straight or branched-chain alkyl        group or the C₁-C₆ straight or branched-chain alkyl group        comprising a phenyl group is unsubstituted or is methyl-,        hydroxyl- or halo-substituted, for example, and without        limitation, R₁, R₂, R₅, and R₆ are independently methyl-,        hydroxyl- or fluoro-substituted, including: methyl, ethyl,        propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,        hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl, or hydroxyphenyl;    -   R₄ is hydrogen, a halo, a C₁-C₆ straight or branched-chain        alkyl, or a C₁-C₆ straight or branched-chain alkyl further        comprising a phenyl (C₆H₅) group, wherein the C₁-C₆ straight or        branched-chain alkyl group or the C₁-C₆ straight or        branched-chain alkyl group comprising a phenyl group is        unsubstituted or is methyl-, hydroxyl- or halo-substituted;    -   R₇ is —C(O)—R₁₃, —C(O)O—R₁₃, or —P(O)—(R₁₃)₂, wherein R₂₄ is        C₁-C₆ straight or branched-chain alkyl or a C₁-C₆ straight or        branched-chain alkyl optionally comprising one or more (C₆H₅)        groups that are independently unsubstituted, or methyl-, ethyl-,        hydroxyl-, halo-substituted or fluoro-substituted, for example        and without limitation, R₇ is Ac (Acetyl, R=—C(O)—CH₃), Boc        (R=—C(O)O-tert-butyl), Cbz (R=—C(O)O-benzyl (Bn)), or a        diphenylphosphate group;    -   R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, where        R₉ is a PARP inhibitor or a derivative or isostere thereof, and        where L is a C₁₋₅ alkyl linker, optionally comprising an ester        or amide linkage;    -   R₃ is a halo, a C₁-C₆ straight or branched-chain alkyl or a        C₁-C₆ straight or branched-chain alkyl further comprising one or        more (C₆H₅) groups that are independently unsubstituted, or        methyl-, ethyl-, hydroxyl- or halo-substituted; and    -   R₁₀, R₁₁, and R₁₂ are independently H or a halo;

-   -   wherein X is

-   -   R₁, R₂, R₅, R₆, and R₁₄ are each independently hydrogen, halo, a        C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆ straight or        branched-chain alkyl further comprising a phenyl (C₆H₅) group,        wherein the C₁-C₆ straight or branched-chain alkyl group or the        C₁-C₆ straight or branched-chain alkyl group comprising a phenyl        group is unsubstituted or is methyl-, hydroxyl- or        halo-substituted;    -   R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉, where        R₉ is a PARP inhibitor or a derivative or isostere thereof, and        where L is a C₁-5 alkyl linker, optionally comprising an ester        or amide linkage; and    -   R₇ is —C(O)—R₁₃, —C(O)O—R₁₃, or —P(O)—(R₁₃)₂, wherein R₁₃ is        C₁-C₆ straight or branched-chain alkyl or a C₁-C₆ straight or        branched-chain alkyl optionally comprising one or more (C₆H₅)        groups that are independently unsubstituted, or methyl-, ethyl-,        hydroxyl-, halo-substituted or fluoro-substituted, for example        and without limitation, R₁₃ is Ac (Acetyl, R=—C(O)—CH₃), Boc        (R=—C(O)O-tert-butyl), Cbz (R=—C(O)O-benzyl (Bn)), or a        diphenylphosphate group; or

-   -   wherein R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or        —O-L-R₉, where R₉ is a PARP inhibitor or a derivative or        isostere thereof, and where L is a C₁₋₅ alkyl linker, optionally        comprising an ester or amide linkage; R₁₅ and R₁₆, independently        are an amine protecting group or acylated. In one aspect, R₁₅        and R₁₆ are protecting groups independently selected from the        group consisting of: 9-fluorenylmethyloxy carbonyl (Fmoc),        t-butyloxycarbonyl (Boc), benzhydryloxycarbonyl (Bhoc),        benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc),        benzyl (Bn), allyloxycarbonyl (alloc), trityl (Trt),        I-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde),        diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts),        dimethoxytrityl (DMT) and monomethoxytrityl (MMT), and R₁₇ is H        or methyl, optionally, R₁₅ is Boc and R₁₆ is Cbz.        11. The compound of clause 10, having the structure of (I) or        (II).        12. The compound of clause 10, having the structure of (III) or        (IV).        13. The compound of clause 10, having the structure of (V).        14. The compound of clause 1, having the structure:

-   -   wherein R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or        —O-L-R₉, where R₉ is a PARP inhibitor, or a derivative or        isostere thereof, and where L is a C₁₋₅ alkyl linker, optionally        comprising an ester or amide linkage.        15. The compound of clause 1, having the structure:

-   -   an isostere thereof, or a pharmaceutically acceptable salt or        ester thereof.        16. The compound of any of clauses 10-15, wherein the PARP        inhibitor is olaparib, veliparib, CEP-8983        (II-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)        or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016        (10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),        INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),        niraparib, talazoparib (BMN673), NU1025        (8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso        quinoline, 4-amino-1,8-naphthalimide, 2-nitro-6[5H]        phenanthridinone, PD128763, and analogues, isosteres, and        derivatives thereof.        17. The compound of any one of clauses 10-16, wherein the PARP        inhibitor is veliparib.        18. The compound of any one of clauses 10-17, wherein R₈ is:

19. A composition comprising a first compound according to any one ofclauses 1-18, and a pharmaceutically-acceptable excipient.20. The composition of clause 19, further comprising a chemotherapeuticagent that is different from the first compound.21. The composition of clause 20, wherein the chemotherapeutic agent isselected from: abiraterone acetate, altretamine, amsacrine, anhydrovinblastine, auristatin, bafetinib, bexarotene, bicalutamide, BMS184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, bleomycin, bosutinib, busulfan, cachectin, cemadotin,chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol,doxetaxel, carboplatin, carmustine (BCNU), chlorambucil, cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, etoposide phosphate, 5-fluorouracil,finasteride, flutamide, hydroxyurea, hydroxyureataxanes, ifosfamide,imatinib, irinotecan, liarozole, lonidamine, lomustine (CCNU), MDV3100,mechlorethamine (nitrogen mustard), melphalan, mitoxantrone, mivobulinisethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,nilotinib, nilutamide, onapristone, oxaliplatin, paclitaxel, ponatinib,prednimustine, procarbazine, RPRI 09881, stramustine phosphate,tamoxifen, tasonermin, taxol, teniposide, topotecan, tretinoin,vinblastine, vincristine, vindesine sulfate, vinflunine, andcombinations thereof, and pharmaceutically acceptable salts or estersthereof.22. A method of treating a cancer in a patient, comprising administeringto a patient an amount of a first compound according to any one ofclauses 1-18 effective to sensitize malignant, but not non-malignant,cells of a patient to anti-cancer drugs.23. The method of clause 23, further comprising administering aradiation therapy to the patient while the first compound is present inthe patient.24. The method of clause 22 or clause 23, further comprisingadministering a chemotherapeutic agent that differs from the firstcompound.25. The method of clause 24, wherein the chemotherapeutic agent isselected from: abiraterone acetate, altretamine, amsacrine, anhydrovinblastine, auristatin, bafetinib, bexarotene, bicalutamide, BMS184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, bleomycin, bosutinib, busulfan, cachectin, cemadotin,chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol,doxetaxel, carboplatin, carmustine (BCNU), chlorambucil, cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, etoposide phosphate, 5-fluorouracil,finasteride, flutamide, hydroxyurea, hydroxyureataxanes, ifosfamide,imatinib, irinotecan, liarozole, lonidamine, lomustine (CCNU), MDV3100,mechlorethamine (nitrogen mustard), melphalan, mitoxantrone, mivobulinisethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,nilotinib, nilutamide, onapristone, oxaliplatin, paclitaxel, ponatinib,prednimustine, procarbazine, RPRI 09881, stramustine phosphate,tamoxifen, tasonermin, taxol, teniposide, topotecan, tretinoin,vinblastine, vincristine, vindesine sulfate, vinflunine, andcombinations thereof, and pharmaceutically acceptable salts or estersthereof.26. A method of reducing NAD⁺ depletion and cell death induced byoxidative stress in a cell or a patient, comprising administering to acell or a patient an amount of a compound according to any one ofclauses 1-18 effective to decrease NAD⁺ depletion in mitochondria of acell or of a patient.27. A method of reducing cell death induced by mitochondrial dysfunctionand/or damage in a cell or a patient, comprising administering to a cellor a patient an amount of a compound according to any one of clauses1-18 effective to improve mitochondrial function and reducemitochondrial damage in a cell or in a patient.28. A method of reducing energy failure induced by ischemia-reperfusionin a cell or a patient, comprising administering to a cell or a patientan amount of a compound according to any one of clauses 1-18 effectiveto prevent or reduce ischemia-reperfusion injury in a cell or in apatient.29. A method of reducing irradiation (IR)-induced cell death andmitochondrial DNA (mtDNA) damage from exposure to ionizing radiation ina patient, comprising administering to a patient an amount of a compoundaccording to any one of clauses 1-18 effective to reduce irradiation(IR)-induced cell death and mitochondrial DNA (mtDNA) damage in apatient.30. Use of a compound according to any one of clauses 1-18 for thepreparation of a medicament for sensitizing malignant, but notnon-malignant cells of a patient to anti-cancer drugs.31. Use of a compound according to any one of clauses 1-18 for thepreparation of a medicament for reducing NAD⁺ depletion and cell deathinduced by oxidative stress in a cell or a patient.32. Use of a compound according to any one of clauses 1-18 for thepreparation of a medicament for reducing cell death induced bymitochondrial dysfunction and/or damage in a cell or a patient.33. Use of a compound according to any one of clauses 1-18 for thepreparation of a medicament for reducing energy failure induced byischemia-reperfusion in a cell or a patient.34. Use of a compound according to any one of clauses 1-18 for thepreparation of a medicament for reducing irradiation (IR)-induced celldeath and mitochondrial DNA (mtDNA) damage from exposure to ionizingradiation in a patient.

The present invention has been described with reference to certainexemplary embodiments, dispersible compositions and uses thereof.However, it will be recognized by those of ordinary skill in the artthat various substitutions, modifications or combinations of any of theexemplary embodiments may be made without departing from the spirit andscope of the invention. Thus, the invention is not limited by thedescription of the exemplary embodiments, but rather by the appendedclaims as originally filed.

We claim:
 1. A compound comprising a mitochondria-targeting gramicidin Speptide isostere moiety covalently linked to a PARP inhibitor moiety, ora pharmaceutically-acceptable salt thereof.
 2. The compound of claim 1,wherein the gramicidin S peptide isostere moiety is an (E)-alkeneisostere of Leu-^(D)Phe-Pro-Val-Orn.
 3. The compound of claim 2, havingthe structure:

Wherein: R₈ is —NH—R₉, —O—R₉, —CH₂—R₉, -L-R₉, —NH-L-R₉, or —O-L-R₉,where R₉ is a PARP inhibitor moiety and L is a C₁₋₅ alkyl linker,optionally comprising an ester or amide linkage; and R₁₇ is methyl orhydrogen, or a pharmaceutically acceptable salt or ester thereof.
 4. Thecompound of claim 3, wherein R17 is hydrogen.
 5. The compound of claim1, wherein the PARP inhibitor moiety is an olaparib, veliparib, CEP-8983(11-methoxy-4,5,6,7-tetrahydro-IH-cyclopenta[a]pyrrolo[3,4-c]carbazole-I,3(2H)-dione)or a prodrug thereof (e.g. CEP-9722), rucaparib, E7016(10-((4-hydroxypiperidin-I-yl)methyl)chromeno-[4,3,2-de]phthalazin-3(2H)-one),INO-1001 (4-phenoxy-3-pyrrolidin-I-yl-5-sulfamoyl-benzoic acid),niraparib, talazoparib (BMN673), NU1025(8-hydroxy-2-methylquinazolin-4(3H)-one), 1,5-dihydroiso quinoline,4-amino-1,8-naphthalimide, 2-nitro-6[5H]phenanthridinone, or PD128763moiety.
 6. The compound of claim 1, wherein the PARP inhibitor isveliparib.
 7. The compound of claim 1, having the structure:

an isostere thereof, or a pharmaceutically acceptable salt or esterthereof.
 8. A composition comprising a first compound according to claim1, and a pharmaceutically-acceptable excipient.
 9. The composition ofclaim 8, further comprising a chemotherapeutic agent that is differentfrom the first compound.
 10. The composition of claim 9, wherein thechemotherapeutic agent is selected from: abiraterone acetate,altretamine, amsacrine, anhydro vinblastine, auristatin, bafetinib,bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin, bosutinib, busulfan, cachectin, cemadotin, chlorambucil,cyclophosphamide, 3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine,docetaxol, doxetaxel, carboplatin, carmustine (BCNU), chlorambucil,cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine(DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, etoposide phosphate, 5-fluorouracil,finasteride, flutamide, hydroxyurea, hydroxyureataxanes, ifosfamide,imatinib, irinotecan, liarozole, lonidamine, lomustine (CCNU), MDV3100,mechlorethamine (nitrogen mustard), melphalan, mitoxantrone, mivobulinisethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,nilotinib, nilutamide, onapristone, oxaliplatin, paclitaxel, ponatinib,prednimustine, procarbazine, RPRI 09881, stramustine phosphate,tamoxifen, tasonermin, taxol, teniposide, topotecan, tretinoin,vinblastine, vincristine, vindesine sulfate, vinflunine, andcombinations thereof, and pharmaceutically acceptable salts or estersthereof.
 11. A method of treating a cancer in a patient, comprisingadministering to a patient an amount of a first compound according toclaim 1 effective to sensitize malignant, but not non-malignant, cellsof a patient to anti-cancer drugs.
 12. The method of claim 11, furthercomprising administering a radiation therapy to the patient while thefirst compound is present in the patient.
 13. The method of claim 11,further comprising administering a chemotherapeutic agent that differsfrom the first compound.
 14. The method of claim 13, wherein thechemotherapeutic agent is selected from: abiraterone acetate,altretamine, amsacrine, anhydro vinblastine, auristatin, bafetinib,bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin, bosutinib, busulfan, cachectin, cemadotin, chlorambucil,cyclophosphamide, 3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine,docetaxol, doxetaxel, carboplatin, carmustine (BCNU), chlorambucil,cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine(DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, etoposide phosphate, 5-fluorouracil,finasteride, flutamide, hydroxyurea, hydroxyureataxanes, ifosfamide,imatinib, irinotecan, liarozole, lonidamine, lomustine (CCNU), MDV3100,mechlorethamine (nitrogen mustard), melphalan, mitoxantrone, mivobulinisethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate,nilotinib, nilutamide, onapristone, oxaliplatin, paclitaxel, ponatinib,prednimustine, procarbazine, RPRI 09881, stramustine phosphate,tamoxifen, tasonermin, taxol, teniposide, topotecan, tretinoin,vinblastine, vincristine, vindesine sulfate, vinflunine, andcombinations thereof, and pharmaceutically acceptable salts or estersthereof.
 15. A method of reducing NAD⁺ depletion and cell death inducedby oxidative stress in a cell or a patient, comprising administering toa cell or a patient an amount of a compound according to claim 1effective to decrease NAD⁺ depletion in mitochondria of a cell or of apatient.
 16. A method of reducing cell death induced by mitochondrialdysfunction and/or damage in a cell or a patient, comprisingadministering to a cell or a patient an amount of a compound accordingto claim 1 effective to improve mitochondrial function and reducemitochondrial damage in a cell or in a patient.
 17. A method of reducingenergy failure induced by ischemia-reperfusion in a cell or a patient,comprising administering to a cell or a patient an amount of a compoundaccording to claim 1 effective to prevent or reduce ischemia-reperfusioninjury in a cell or in a patient.
 18. A method of reducing irradiation(IR)-induced cell death and mitochondrial DNA (mtDNA) damage fromexposure to ionizing radiation in a patient, comprising administering toa patient an amount of a compound according to claim 1 effective toreduce irradiation (IR)-induced cell death and mitochondrial DNA (mtDNA)damage in a patient.